Although it has long been known that increases in neuronal activity evoke localized increases in cerebral blood flow (CBF), the mechanisms involved in this functional hyperemia have not been fully explained. Because CBF is used as a surrogate for mapping human brain function in health and disease, it is critical to understand the neurovascular coupling mechanisms, which could be altered by vascular or neurological diseases. Previous work done in this project demonstrated that astrocytes produce EETs which hyperpolarize and dilate cerebral vascular smooth muscle (VSM) and that inhibitors of this pathway markedly attenuate the increase in CBF evoked by NMDA, whisker or forepaw stimulation. More recently, we found that stimulation of metabotropic glutamate receptors (mGluR) activates a novel Kca channel in astrocytes by an epoxygenase-dependent mechanism. Glutamate also activates Kca channels in VSM via a mechanism that is dependent on the release of EETs by astrocytes. However, the mechanism by which activation of glutamate receptors promote the synthesis and release of EETs by astrocytes is unknown and is one of the major focuses of this proposal. The working hypothesis is that astrocytes couple CBF to neuronal activity through activation of mGluR in astrocytes leading to localized increases in Ca 2+ and synthesis of EETs, which together open Kca channels and lead to astrocyte hyperpolarization and further capacitive Ca 2+ influx. This sequence activates phospholipases and promotes astrocyte release of EETs which, in turn, diffuse to nearby VSM to open Kca channels and produce vasodilation. In Specific Aim 1, the mechanisms by which mGtuR activation promotes the release of EETs through Ca 2+ signaling pathways will be investigated. In Specific Aim 2, the interaction of EETs with other potential vasodilatory mechanisms involving nitric oxide, heine oxygenase, and 2A and 2B adenosine receptors in mediating functional hyperemia will be investigated using both pharmacological and gene knockout strategies. In Specific Aim 3, the effect of increasing EETs availability on functional hyperemia will be examined by reducing EETs breakdown using both pharmacological inhibitors and epoxide hydrolase knockout strain of mice. In Specific Aim 4, the effect of acute and chronic hypoxic exposure on altering the control mechanisms that match blood flow to metabolic activity in the brain will be investigated. Experiments will focus on P450 epoxygenase activity and expression which, in preliminary data, are found to be upregulated by hypoxia. Parallel studies will be performed in cultured astrocytes to determine the effect of hypoxia on the coupling of mGluR activation to Ca 2+ signaling, Kca channel function, and the release of EETs. Therefore, the proposed interdisciplinary studies will offer a more comprehensive view at both the cellular and integrated level on the mechanisms by which CBF is coupled to neuronal activity and how neurotransmitters promote the release of EETs from astrocytes.